Project Summary/Abstract Hospital related bacterial infections which are generally associated with biofilms are a significant health care problem in the US. Although bacteria that repopulate biofilms are often sensitive to the antibiotics used, biofilms interestingly have a high propensity to relapse. In this phenomenon, after the majority of the cells have been killed upon exposure to antibiotics, a small number of tolerant cells survive the treatment. This subpopulation eventually regains their ability to proliferate after the conclusion of treatment, and forms a cell population that has the same antibiotic sensitivity of the original cell population. Persisters and ?viable but non- culturable? cells (VBNCs) are two phenotypic variants identified so far, which are highly tolerant to antibiotics and the underlying cause of this infection reoccurrence. Unfortunately, many physiological aspects of these phenotypes remain elusive and this significant knowledge gap has hindered the discovery of anti-persister and anti-VBNC therapeutics. The overall goal of the proposed research is to identify the molecular-level characteristics that portray persisters and VBNCs, and explore the methods facilitating the discovery of novel therapeutic strategies that eliminate both cell types. In this context, we will first generate a transposon or overexpression library for E. coli and screen for genes whose perturbation would impair both persister and VBNC formation using fluorescence activated cell sorting (FACS) and transposon-sequencing. To further map the underlying mechanisms that are relevant to an antibiotic tolerant state, we will determine transcriptome patterns of the strains which have received genetic perturbations eliminating the antibiotic tolerant states. The same methodology will be applied to an opportunistic pathogen P. aeruginosa to systematically explore its genetic basis of persistence and VBNC state. Altogether, these experiments will provide insights to develop novel approaches to perturb the ability of bacteria to form antibiotic tolerant states. In addition, this study would represent one of the seminal contributions to characterizing and distinguishing persister and VBNC formation mechanisms in P. aeruginosa.